In the life science field, preparation of recombinant proteins is performed as a part of basic research, applied research and product development. However, there are a limited number of techniques for isolating and purifying the proteins in high purity.
Affinity chromatography is one of the most powerful means for protein purification. As an isolation and purification method for proteins using affinity chromatography, a method involving attaching a histidine-containing peptide of 6 to 10 residues (histidine tag) to the N or C terminus of proteins and using the interaction of the histidine tag and a metal such as nickel is known. A method using the interaction of a tag peptide (peptide tag) and an antibody thereagainst is also known (for example, nonpatent literatures 1 and 2).
However, in the former method using the histidine tag, because of low specificity between nickel and the histidine tag, proteins other than objective proteins (histidine tagged proteins), and even compounds other than proteins adsorb onto the column. Therefore, this method has a problem in that highly purified proteins cannot be obtained in a single purification step.
As a detection and purification system for proteins using the interaction of a tag peptide and an antibody thereagainst like the latter method, a FLAG (registered trademark) system commercially available from Sigma is used extensively. This technique, in which a FLAG peptide and an antibody thereagainst (antibodies M1 and M2, etc.) are used, is currently considered the most excellent in specificity. However, the FLAG (registered trademark) system is so expensive that it may be limitedly used in terms of cost.
In conventional techniques using a tag peptide and an antibody thereagainst, the antigen (tag peptide)-antibody interaction is so strong that it is not easy to elute antigens from an immunoaffinity column once the antigens bind to antibodies thereagainst. For this reason, in affinity purification methods for proteins, strong acid (for example, pH 3) or alkaline (for example, pH 10) solutions, protein denaturants (high-concentration urea or guanidine hydrochloride) or the like are usually used as an eluent for antigens. However, the use of these eluents is disadvantageous because they denature or destabilize objective proteins and particularly results in very poor yields of multi-subunit enzymes etc. The use of such eluents has another disadvantage that antibodies used for the purification column cannot be repeatedly used because the antibodies easily deteriorate as well. In the case of the FLAG (registered trademark) system as well, repeated use of antibodies M1 and M2 for purification etc. is limited because of their decline in specificity to the antigen.
Therefore, at this point, no purification system that enables proteins to be isolated and purified in high purity and in an inexpensive and easy manner and can endure repeated use is developed yet.